Arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione as novel multitarget 5-HT/D receptor agents with potential antipsychotic activity

Abstract

A series of new 7-arylpiperazinylalkyl-1,3-dimethyl-purine-2,6-dione derivatives with diversified 8-amino substituent in 8 position was synthesized and their 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptor affinities were determined. The binding study allowed identifying some potent 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands. The most interesting because of their multireceptor profile were 8-piperidine (30–35) and 8-dipropylamine (45–47) analogs with four and five carbon aliphatic linkers. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. The advantageous in vitro affinity of compound 34 for 5-HT_1A and D₂ receptors has been explained by means of molecular modeling, taking into consideration its partial agonist activity towards the latter one. In behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice.

• Antipsychotics
• arylpiperazines
• D₂ partial agonist
• derotonin receptor ligands

Introduction

Development of the atypical antipsychotic drugs (AADs), such as olanzapine, quetiapine, and risperidone and more recently ziprazidone and paliperidone, was crucial innovation for the contemporary pharmacotherapy of schizophrenia. In comparison with the first-generation antipsychotics like, e.g. haloperidol, chlorpromazine, or fluphenazine, AADs interact with many serotoninergic receptors including 5-HT_1A, 5-HT_2A, 5-HT₆, 5HT₇, and dopaminergic D₂ receptors1^,2. The multiple mechanism of their action significantly reduced the range of the observed adverse effects, such as extrapyramidal symptoms3 but those drugs did not eliminate cognitive deficits in schizophrenia. Therefore, new therapeutic agents with dual effects, e.g. suppression of psychotic symptoms and elimination of cognition impairment are still needed4. In the recent years, the new class of antipsychotics called “dopamine stabilizers”, exemplified by aripiprazole and compounds investigated in clinical trials, e.g. brexpiprazole and cariprazine have been identified (Figure 1). Aripiprazole possessing broad spectrum of clinical benefits (antipsychotic, antidepressant, and anxiolytic effects) through unique receptor profile, partial agonist of D₂ and 5-HT₁ receptors and antagonist of 5-HT_2A and 5-HT₇ sites, was revealed to be effective against schizophrenia with anxiogenic and depressive symptoms. Moreover, aripiprazole received supplemental approval for the treatment of the acute manic episodes of bipolar disorder and as an adjunct therapy for major depressive disorder5. The new class of dopamine stabilizers represents an important therapeutic innovation in psychopharmacology and is distinct from D₂ antagonists in terms of side effects and improvement of negative and cognitive symptoms. Several series of aripiprazole analogs, e.g. quinoline- and isoquinoline – amide and sulfonamide – derivatives of long-chain arylpiperazines (LCAPs) have been explored as potential antipsychotic, antidepressant, and anxiolytic agents (Figure 1)6–8.

Figure 1. The structures of representatives of dopamine stabilizers.

Among the currently numerous pharmacological approaches for schizophrenia treatments, considerable attention has been focused on 5-HT₆ antagonists. Because of their distribution in cerebral cortex and limbic areas, 5-HT₆ receptors were proposed to be involved in cognitive process. This modulatory activity including elevating extracellular levels of both glutamate and acetylcholine suggests potential utility for 5-HT₆ receptor antagonist in the treatment of cognitive impairments associated with schizophrenia9–11. Taking into account the above-mentioned findings, the multitarget strategy seems to be one of the most valuable approaches in the development of CNS active agents with potential antipsychotic, anxiolytic, and/or antidepressant-like activities.

For several years, our attention has been focused on the development of agents generally classified as long-chain arylpiperazines (LCAPs), containing 1,3-dimethylpurine-2,6-dione system as a terminal amide fragment, which were mainly evaluated toward 5-HT_1A, 5-HT_2A, and 5-HT₇ receptors12–18 and recently also for 5-HT₆ and D₂ receptors19^,20. Among them, several compounds containing 8-alkoxy- or 8-morpholinyl-1,3-dimethylpurine-2,6-dione moiety showed features of a 5-HT_1A/5-HT₇ or 5-HT_1A/5-HT_2A receptor ligand and produced antidepressant- and anxiolytic-like properties in behavioral tests in mice15^,18^,21. The multireceptor strategy was also explored among 8-unsubstituted 1,3-dimethylpurine-2,6-dione derivatives of LCAPs19. The study allowed us to identify some potent 5-HT_1A receptor ligands with additional moderate affinity for 5-HT_2A, 5-HT₇, and D₂ receptors and a diversified 5-HT_1A receptor functional profile which showed antidepressant- and anxiolytic-like activities even greater than those of the reference compounds (imipramine and diazepam, respectively)19.

Continuing our study in a group of purine-2,6-dione derivatives of LCAPs and to extend the studies aimed at verification of the kind of substituent in a 8 position of the purine-2,6-dione system on the selected serotonin and dopamine receptor affinity, we designed a novel series of 7-arylpiperazinylalkyl-8-amino-1,3-dimethylpurine-2,6-dione derivatives (18–47). The influence of three structure features – the kind of amine moiety in the 8-position of purine-2,6-dione system, the linker length between the purine-2,6-dione core and phenylpiperazine (PhP) fragment, and the kind of substituent in the phenyl ring were explored.

Herein, we report on their synthesis and in vitro affinity evaluation for selected 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptors, as well as determination of their intrinsic activity. Moreover, the potential antipsychotic-like properties of the selected compounds 32 and 34 in the behavioral test in mice will be presented. This is interesting in terms of validation of the multireceptor profile of the designed compounds which can lead to the formation of potential psychotropic (antipsychotic, antidepressant, and anxiolytic) agents.

Methods

Chemistry

Organic solvents (from Sigma-Aldrich, St. Louis, MO and Chempur, Karlsruhe, Germany) were of reagent grade and were used without purification. All other reagents were from Sigma-Aldrich (St. Louis, MO) and Alfa Aesar. Melting points (m.p.) were determined with a Büchi apparatus (BUCHI Labortechnik, Essen, Germany) and are uncorrected. All the compounds were routinely checked by TLC using Merck Kieselgel 60 F₂₅₄ sheets (Merck, Darmstadt, Germany) with the following solvent: (A) dichloromethane/methanol (9.5:0.5). Spots were detected by UV absorption. Column chromatography separations were carried out on column with Merck Kieselgel 60 (Merck, Darmstadt, Germany) using the solvent: (A) dichloromethane/methanol (9.5:0.5). ¹H-NMR spectra were taken with a Varian Mercury-VX (300 MHz) spectrometer (Merck, Darmstadt, Germany) in CDCl₃ (6 and 10–17) or DMSO-d₆ (18–47) solutions, using signal of solvent’s residual ¹H atoms as an internal standard (δ = 7.26 ppm and 2.48 ppm, respectively). Chemical shifts were expressed in δ (ppm) and the coupling constants J in Hertz (Hz) and splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).

LC/MS analysis was performed on Waters Acquity TQD apparatus (Waters Corporation, Milford, MA) with eλ DAD detector. For mass spectrometry ESI + (electrospray positive), ionization mode was used. UV spectra were taken in the range of 200–700 nm. For establishing the purity of compounds, UV chromatograms were used. The UPLC/MS purity of all the investigated compounds was determined to be over 98%. Elemental analysis was taken with Elementar Vario EL III apparatus. All analyses were within ± 0.4% of the theoretical values.

The synthesis of the designed 8-amino-7-arylpiperazinylalkyl-1,3-dimethylpurine-2,6-diones hydrochlorides (18–47) is presented in the Scheme 1.

Scheme 1. The synthesis of 8-amino-purine-2,6-dione derivatives of LCAP 18–47. Reagents and conditions: (a) 1-chloro-3-bromopropane, 1-chloro-4-bromobutane, or 1-chloro-5-bromopentane, K₂CO₃, TEBA, acetone, boiling temp., 15–17 h; (b) 1-arylpiperazine, K₂CO₃, 1-propanol, 80°C, (c) conc. HCl, acetone.

The starting 8-bromo-1,3-dimethylpurine-2,6-dione (1) was synthesized by a procedure published elsewhere22. The 1,3-dimethyl-8-pyrrolidin-1-yl-7H-purine-2,6-dione (2)23, 1,3-dimethyl-8-(1-piperidyl)-7H-purine-2,6-dione (3)23, 1,3-dimethyl-8-morpholino-7H-purine-2,6-dione (4)23, 8-(diethylamino)-1,3-dimethyl-7H-purine-2,6-dione (5)24, and new 8-(dipropylamino)-1,3-dimethyl-7H-purine-2,6-dione (6) were obtained from 1 according to the previously described method23.

8-(Dipropylamino)-1,3-dimethyl-7H-purine-2,6-dione (6)

Yield: 75%, m.p. 188–190 °C, R_f = 0.51 (A), ¹H-NMR (CDCl₃) δ: 0.92 (t, J = 7.4 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.56–1.74 (m, 4H, N(CH₂CH₂CH₃)₂), 3.40 (s, 3H, N1-CH₃), 3.44–3.52 (m, 4H, N(CH₂CH₂CH₃)₂), 3.55 (s, 3H, N3-CH₃), 10.55 (s, 1H, N7-H). LC/MS: m/z calc. 280.17, found 280.37. Anal. calcd. for C₁₃H₂₁N₅O₂: C, H, N.

The 7-(3-chloropropyl)-1,3-dimethyl-8-(1-piperidyl)purine-2,6-dione (7)25, 7-(4-chlorobutyl)-1,3-dimethyl-8-morpholino-purine-2,6-dione (8)18, 7-(3-chloropropyl)-8-(diethylamino)-1,3-dimethylpurine-2,6-dione (9)25, and new 1,3-dimethyl-7-chloroalkyl-8-amino-purine-2,6-dione derivatives (10–17) were prepared in a reaction of 10 mmol of 2–6 with 20 mmol appropriate alkylating agent (1-bromo-3-chloropropane, 1-bromo-4-chlorobutane, and 1-bromo-5-chloropentane) according to the previously described method26. Compounds 7–17 were purified by crystallization from methanol (Scheme 1).

7-(3-Chloropropyl)-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione (10)

Yield 74%, m.p. 129–130 °C, R_f = 0.59 (A), ¹H-NMR (CDCl₃) δ: 1.98–2.03 (m, 4H, 3,4-pyrrolidine), 2.21–2.30 (m, 2H, N7-CH₂CH₂CH₂Cl), 3.37 (s, 3H, N1-CH₃), 3.55–3.59 (m, 2H, CH₂Cl), 3.57 (s, 3H, N3-CH₃), 3.65–3.68 (m, 4H, 2,5-pyrrolidine), 4.38–4.43 (m, 2H, N7-CH₂CH₂CH₂Cl). LC/MS: m/z calc. 326.13, found 326.30. Anal. calcd. for C₁₄H₂₀ClN₅O₂: C, H, N.

7-(4-Chlorobutyl)-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione (11)

Yield 62%, m.p. 97–99 °C, R_f = 0.58 (A), ¹H-NMR (CDCl₃) δ: 1.71–1.82 (m, 2H, N7-CH₂CH₂(CH₂)₂), 1.83–1.94 (m, 2H, N7-(CH₂)₂CH₂CH₂), 1.99–2.20 (m, 4 H, 3,4-pyrrolidine), 3.37 (s, 3H, N1-CH₃), 3.51 (s, 3H, N3-CH₃), 3.56 (m, 6H, 2,5-pyrrolidine & N7-(CH₂)₂CH₂CH₂Cl), 4.28 (t, J = 7.3 Hz, 2H, N7-CH₂CH₂(CH₂)₂). LC/MS: m/z calc. 340.15, found 340.33. Anal. calcd. for C₁₅H₂₂ClN₅O₂: C, H, N.

7-(4-Chlorobutyl)-1,3-dimethyl-8-(1-piperidyl)purine-2,6-dione (12)

Yield 70%, m.p. 101–103 °C, R_f = 0.58 (A), ¹H-NMR (CDCl₃) δ: 1.57–1.78 (m, 6H, (CH₂)₃ piperidine), 1.98–2.05 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.10–3.20 (m, 4H, N(CH₂)₂ piperidine), 3.38 (s, 3H, N1-CH₃), 3.53 (s, 3H, N3-CH₃), 3.56 (t, J = 6.0 Hz, 2H, CH₂Cl), 4.10 (t, J = 7.3 Hz, 2H, N7-CH₂). LC/MS: m/z calc. 354.16, found 354.06. Anal. calcd. for C₁₆H₂₄ClN₅O₂: C, H, N.

7-(5-Chloropentyl)-1,3-dimethyl-8-(1-piperidyl)purine-2,6-dione (13)

Yield 69%, m.p. 72–73 °C, R_f = 0.66 (A), ¹H-NMR (DMSO) δ: 1.38–1.48 (m, 2H, N7-(CH₂)₂CH₂(CH₂)₂), 1.63–1.78 (m, 6H, (CH₂)₃ piperidine), 1.82–1.88 (m, 4H, N7-CH₂CH₂CH₂CH₂) 3.10–3.20 (m, 4H, N(CH₂)₂ piperidine), 3.38 (s, 3H, N1-CH₃), 3.53 (s, 3H, N3-CH₃), 3.55 (t, J = 6.4 Hz, 2H, CH₂Cl), 4.05 (t, J = 7.3 Hz, 2H, N7-CH₂). LC/MS: m/z calc. 368.18, found 368.03. Anal. calcd. for C₁₇H₂₆ClN₅O₂: C, H, N.

7-(5-Chloropentyl)-1,3-dimethyl-8-morpholino-purine-2,6-dione (14)

Yield 75%, m.p. 87–88 °C, R_f = 0.58 (A), ¹H-NMR (CDCl₃) δ: 1.37–1.49 (m, 2H, N7-(CH₂)₂CH₂(CH₂)₂), 1.84–1.93 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.19–3.25 (m, 4H, N(CH₂CH₂)₂O), 3.39 (s, 3H, N1-CH₃), 3.49–3.59 (m, 5H, N3-CH₃, CH₂Cl), 3.81–3.89 (m, 4H, N(CH₂CH₂)₂O), 4.12 (t, J = 7.1 Hz, 2H, N7-CH₂). LC/MS: m/z calc. 370.16, found 370.38. Anal. calcd. for C₁₆H₂₄ClN₅O₃: C, H, N.

7-(4-Chlorobutyl)-8-(diethylamino)-1,3-dimethyl-purine-2,6-dione (15)

Yield 65%, m.p. 84–86 °C, R_f = 0.71 (A), ¹H-NMR (CDCl₃) δ: 1.14 (t, J = 7.1 Hz, 6H, N(CH₂CH₃)₂), 1.62–1.78 (m, 2H, N7-(CH₂)₂CH₂CH₂), 1.91–1.98 (m, 2H, N7-CH₂CH₂(CH₂)₂), 3.26 (q, J = 7.1 Hz, 4H, N(CH₂CH₃)₂), 3.39 (s, 3H, N1-CH₃), 3.47–3.56 (m, 5H, N3-CH₃, CH₂Cl), 4.25 (t, J = 7.1 Hz, 2H, N7-CH₂). LC/MS: m/z calc. 342.16, found 342.39. Anal. calcd. for C₁₅H₂₄ClN₅O₂: C, H, N.

7-(3-Chloropropyl)-8-(dipropyloamino)-1,3-dimethyl-purine-2,6-dione (16)

Yield 73%, m.p. 88–90 °C, R_f = 0.45 (A), ¹H-NMR (CDCl₃) δ: 0.90 (t, J = 7.1 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.52–1.65 (m, 4H, N(CH₂CH₂CH₃)₂), 2.25–2.34 (m, 2H, N7-CH₂CH₂CH₂), 3.16–3.21 (m, 4H, N(CH₂CH₂CH₃)₂), 3.37 (s, 3H, N1-CH₃), 3.52 (s, 3H, N3-CH₃), 3.54–3.58 (t, J = 6.1 Hz, 2H, CH₂Cl), 4.25 (t, J = 7,4 Hz, 2H, N7-CH₂(CH₂)₂). LC/MS: m/z calc. 356.18, found 356.42. Anal. calcd. for C₁₆H₂₆ClN₅O₂: C, H, N.

7-(4-Chlorobutyl)-8-(dipropyloamino)-1,3-dimethyl-purine-2,6-dione (17)

Yield 71%, m.p. 38–40 °C, R_f = 0.58 (A), ¹H-NMR (CDCl₃) δ: 0.89 (t, J = 7.4 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.52–1.62 (m, 4H, N(CH₂CH₂CH₃)₂), 1.70–1.79 (m, 2H, N7-(CH₂)₂CH₂CH₂), 1.90–1.98 (m, 2H, N7-CH₂CH₂(CH₂)₂), 3.14–3.19 (m, 4H, N(CH₂CH₂CH₃)₂), 3.37 (s, 3H, N1-CH₃), 3.51 (s, 3H, N3-CH₃), 3.54–3.56 (m, 2H, CH₂Cl), 4.14 (t, J = 7.4 Hz, 2H, N7-CH₂(CH₂)₃). LC/MS: m/z calc. 370.20, found 370.44. Anal. calcd. for C₁₇H₂₈ClN₅O₃: C, H, N.

General procedure for the preparation of 8-amino-7-arylpiperazinylalkyl-1,3-dimethylpurine-2,6-diones hydrochlorides (18–47)

A mixture of the appropriate 8-amino-7-chloroalkyl-1,3-dimethylpurine-2,6-dione derivatives 7–17 (10 mmol), respective 1-phenylpiperazine derivatives (1-phenylpiperazine, 1–(2-methoxyphenyl)piperazine, 1–(3-chlorophenyl)piperazine, 1–(2-fluorophenyl)piperazine, 1–(4-fluorophenyl)piperazine, 1–(3,4-dichlorophenyl)-piperazine (10 mmol), and anhydrous K₂CO₃ (20 mmol) were refluxed in 1-propanol (10 mL) for 40 h. Then the mixture was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in a small amount of acetone, acidified with conc. HCl to pH = 3 and cooled. The precipitate hydrochloride salt was filtered off and purified by crystallization from ethanol.

1,3-Dimethyl-7-[3–(4-phenylpiperazin-1-yl)propyl]-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (18)

Yield 62%, m.p. 157–159 °C, R_f = 0.49 (A), ¹H-NMR (DMSO) δ: 1.21–1.26 (m, 2H, N7-(CH₂)₂CH₂(CH₂)₂), 1.38 (t, J = 7.1 Hz, 3H, O-CH₂CH₃) 1.60–1.80 (m, 4H, N7-CH₂CH₂CH₂CH₂CH₂), 2.82–3.18 (m, 6H, (CH₂)₂N-Ph & N7-(CH₂)₃CH₂CH₂), 3.20 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.40–3.58 (m, 2H, N(CH₂)₂), 3.76 (s, 3H, Ph-OCH₃), 4.00 (t, J = 6.5 Hz 2H, N7-CH₂CH₂), 4.50 (q, J = 7.0 Hz, 2H, O-CH₂CH₃), 6.82–7.02 (m, 4H, Ph), 10.77 (s, 1H, H⁺). LC/MS: m/z calc. 452.27, found 452.50. Anal. calcd. for C₂₄H₃₃N₇O₂···HCl: C, H, N.

7-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (19)

Yield 46%, m.p. 241–243 °C, R_f = 0.20 (A), ¹H-NMR (DMSO) δ: 1.86–1.98 (m, 4H, 3,4-pyrrolidine), 2.11–2.22 (m, 2H, N7-CH₂CH₂CH₂), 2.98–3.14 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.34 (s, 3H, N3-CH₃), 3.48–3.60 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.63–3.76 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.77 (s, 3H, OCH₃), 4.26 (t, J = 7.1 Hz, 2H, N7-CH₂), 6.86–7.04 m, 4H, Ph), 10.84 (s, 1H, NH⁺). LC/MS: m/z calc. 482.28, found 482.34. Anal. calcd. for C₂₅H₃₅N₇O₂···HCl: C, H, N.

7-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (20)

Yield 53%, m.p. 237–240 °C, R_f = 0.26 (A), ¹H-NMR (DMSO) δ: 1.85–1.97 (m, 4H, 3,4-pyrrolidine), 2.09–2.22 (m, 2H, N7-CH₂CH₂CH₂), 2.96–3.15 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.46–3.62 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.65–3.78 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.25 (t, J = 7.1 Hz, 2H, N7-CH₂), 6.85 (dd, ³J = 7.7 Hz, ⁴J = 1.8 Hz, 1H, Ph), 6.94 (dd, ³J = 8.4 Hz, ⁴J = 1.9 Hz, 1H, Ph), 7.03 (t, J = 2.2 Hz, 1H, Ph), 7.24 (t, J = 8.2 Hz, 1H, Ph), 10.86 (s, 1H, NH⁺). LC/MS: m/z calc. 486.23, found 486.40. Anal. calcd. for C₂₄H₃₂N₇O₂···HCl: C, H, N.

7-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (21)

Yield 48%, m.p. 248–250 °C, R_f = 0.34 (A), ¹H-NMR (DMSO) δ: 1.84–1.97 (m, 4H, 3,4-pyrrolidine), 2.10–2.20 (m, 2H, N7-CH₂CH₂CH₂), 2.96–3.15 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.34 (s, 3H, N3-CH₃), 3.49–3.61 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.65–3.75 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.27 (t, J = 7.0 Hz, 2H, N7-CH₂), 6.94–7.03 (m, 2H, Ph), 7.03–7.13 (m, 2H, Ph), 10.90 (s, 1H, NH⁺). LC/MS: m/z calc. 470.26, found 470.49. Anal. calcd. for C₂₄H₃₂N₇O₂···HCl: C, H, N.

1,3-Dimethyl-7-[4-(4-phenylpiperazin-1-yl)butyl]-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (22)

Yield 53%, m.p. 208–211 °C, R_f = 0.31 (A), ¹H-NMR (DMSO) δ: 1.59–1.76 (m, 4H, N7-CH₂CH₂CH₂CH₂), 1.83–1.97 (m, 4H, 3,4-pyrrolidine), 2.98–3.24 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.17 (s, 3H, N1-CH₃), 3.34 (s, 3H, N3-CH₃), 3.41–3.60 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.71–3.82 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.28 (m, 2H, N7-CH₂), 6.84 (t, J = 7.3 Hz, 1H, Ph), 6.93–7.03 (d, J = 8.0 Hz, 2H, Ph), 7.18–7.31 (m, 2H, Ph), 10.96 (s, 1H, NH⁺). LC/MS: m/z calc. 466.29, found 466.57. Anal. calcd. for C₂₅H₃₅N₇O₂···HCl: C, H, N.

7-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (23)

Yield 49%, m.p. 177–179 °C, R_f = 0.14 (A), ¹H-NMR (DMSO) δ: 1.58–1.75 (m, 4H, N7-CH₂CH₂CH₂CH₂), 1.85–1.95 (m, 4H, 3,4-pyrrolidine), 2.96–3.18 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.34 (s, 3H, N3-CH₃), 3.48–3.60 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.68–3.80 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.76 (s, 3H, OCH₃), 4.28 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.86–7.02 (m, 4H, Ph), 10.94 (s, 1H, NH⁺). LC/MS: m/z calc. 496.30, found 496.20. Anal. calcd. for C₂₆H₃₇N₇O₃···HCl: C, H, N.

7-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (24)

Yield 51%, m.p. 226–228 °C, R_f = 0.23 (A), ¹H-NMR (DMSO) δ: 1.60–1.77 (m, 4H, N7-CH₂CH₂CH₂CH₂), 1.90–1.93 (m, 4H, 3,4-pyrrolidine), 2.98–3.18 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.34 (s, 3H, N3-CH₃), 3.45–3.58 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.71–3.80 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.27 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.86 (d, J = 7.6 Hz, 1H, Ph), 6.95 (d, J = 8.4 Hz, 1H, Ph), 7.04 (s, 1H, Ph), 7.24 (t, J = 8.0 Hz, 1H, Ph), 10.96 (s, 1H, NH⁺). LC/MS: m/z calc. 500.25, found 500.43. Anal. calcd. for C₂₅H₃₄ClN₇O₂···HCl: C, H, N.

7-{4-[4-(4-Fluorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-pyrrolidin-1-yl-purine-2,6-dione hydrochloride (25)

Yield 58%, m.p. 211–213 °C, R_f = 0.31 (A), ¹H-NMR (DMSO) δ: 1.70–1.80 (m, 4H, N7-CH₂CH₂CH₂CH₂), 1.90–1.92 (m, 4H, 3,4-pyrrolidine), 3.01–3.18 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.18 (s, 3H, N1-CH₃), 3.35 (s, 3H, N3-CH₃), 3.46–3.56 (m, 6H, 2,5-pyrrolidine, NH⁺(CH₂CH₂)₂N), 3.71–3.82 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.22 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.98–7.12 (m, 4H, Ph), 10.61 (s, 1H, NH⁺). LC/MS: m/z calc. 484.28, found 484.40. Anal. calcd. for C₂₅H₃₄FN₇O₂···HCl: C, H, N.

1,3-Dimethyl-7-[3–(4-phenylpiperazin-1-yl)propyl]-8-(1-piperidyl)-purine-2,6-dione hydrochloride (26)

Yield 58%, m.p. 243–245°C, R_f = 0.45 (A), ¹H-NMR (DMSO-d₆) δ: 1.51–1.72 (m, 6H, (CH₂)₃ piperidine), 2.16–2.27 (m, 2H, N7-CH₂CH₂CH₂), 3.02–3.17 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.20 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.48–3.57 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.77–3.81 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.11 (t, ³J = 6.8 Hz, 2H, N7-CH₂), 6.84 (t, J = 7.1 Hz, 1H, Ph), 6.97 (d, J = 8.0 Hz, 2H, Ph), 7.20–7.28 (m, 2H, Ph), 10.50 (s, 1H NH⁺). LC/MS: m/z calc. 466.29, found 466.02. Anal. calcd. for C₂₅H₃₅N₇O₂···HCl: C, H, N.

7-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (27)

Yield 67%, m.p. 241–242 °C, R_f = 0.35 (A), ¹H-NMR (DMSO-d₆) δ: 1.52–1.70 (m, 6H, (CH₂)₃ piperidine), 2.17–2.27 (m, 2H, N7-CH₂CH₂CH₂), 2.92–3.18 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.20 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.48–3.52 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.76 (s, 3H, OCH₃), 3.99–4.15 (m, 4H, NH⁺(CH₂CH₂)₂N, N7-CH₂), 6.86–7.03 (m, 4H, Ph), 10.57 (s, 1H NH⁺). LC/MS: m/z calc. 496.30, found 496.55. Anal. calcd. for C₂₆H₃₇N₇O₃···HCl: C, H, N.

7-{3-[4-(3,4-Dichlorophenyl)piperazin-1-yl]propyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (28)

Yield 62%, m.p. 252–254 °C, R_f = 0.40 (A), ¹H-NMR (DMSO-d₆) δ: 1.51–1.69 (m, 6H, (CH₂)₃ piperidine), 2.12–2.23 (m, 2H, N7-CH₂CH₂CH₂), 3.01–3.17 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.20 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.48–3.55 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.88–3.91 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.09 (t, J = 7.4 Hz, 2H, N7-CH₂), 6.97–7.03 (m 1H, Ph), 7.20–7.25 (m, 1H, Ph), 7.42–7.47 (d, J = 8.7 Hz, 1H, Ph), 10.14 (s, 1H NH⁺). LC/MS: m/z calc. 534.21, found 533.90. Anal. calcd. for C₂₅H₃₃Cl₂N₇O₂···HCl: C, H, N.

1,3-Dimethyl-7-[4-(4-phenylpiperazin-1-yl)butyl]-8-(1-piperidyl)-purine-2,6-dione hydrochloride (29)

Yield 57%, m.p. 200–201 °C, R_f = 0.34 (A), ¹H-NMR (DMSO) δ: 1.58–1.65 (m, 8H, N7-(CH₂)₂CH₂ (CH₂)₃, piperidine), 1.69–1.78 (m, 2H, N7-CH₂CH₂), 3.02–3.18 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.49–3.55 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 3.78–3.85 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 4.08 (t, J = 7.0 Hz, 2H, N7-CH₂), 6.84 (t, J = 7.3 Hz, 1H, Ph), 6.98 (d, J = 8.0 Hz, 2H, Ph), 7.23 (t, J = 8.0 Hz, 2H, Ph), 10.22 (s, 1H, NH⁺). LC/MS: m/z calc. 480.30, found 480.57. Anal. calcd. for C₂₆H₃₇N₇O₂···HCl: C, H, N.

7-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (30)

Yield 71%, m.p. 198–199 °C, R_f = 0.28 (A), ¹H-NMR (DMSO) δ: 1.57–1.78 (m, 10H, (CH₂)₃ piperidine, N7-CH₂CH₂CH₂CH₂), 3.05–3.17 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.44–3.56 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 3.75 (s, 3H, OCH₃), 4.05 (t, J = 6.9 Hz, 2H, N7-CH₂); 6,87–7,00 (m, 4H, Ph). 10.98 (s, 1H, NH⁺). LC/MS: m/z calc. 510.31, found 510.04. Anal. calcd. for C₂₇H₃₉N₇O₃···HCl: C, H, N.

7-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (31)

Yield 58%, m.p. 223–224 °C, R_f = 0.38 (A), ¹H-NMR (DMSO) δ: 1.57–1.65 (m, 8H, (CH₂)₃ piperidine, N7-(CH₂)₂CH₂), 1.67–1.78 (m, 2H, N7CH₂CH₂), 3.05–3.18 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.40–3.45 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 3.80–3.89 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 4.06 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.85 (dd, ³J = 8.0 Hz, ⁴J = 1.7 Hz, 1H, Ph), 6.94 (dd, J = 8.0 Hz, 1H, Ph), 7.03 (s, 1H, Ph), 7.24 (t, J = 8.0 Hz, 1H, Ph), 10.25 (s, 1H, NH⁺). LC/MS: m/z calc. 514.26, found 514.00. Anal. calcd. for C₂₆H₃₆ClN₇O₂···HCl: C, H, N.

7-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (32)

Yield 67%, m.p. 210–211 °C, R_f = 0.32 (A), ¹H-NMR (DMSO) δ: 1.57–1.68 (m, 8H, (CH₂)₃ piperidine, N7-(CH₂)₂CH₂), 1.70–1.77 (m, 2H, N7-CH₂CH₂), 3.06–3.17 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.41–3.50 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 3.80–3.88 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 4.06 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.96 (dd, ³J = 8.1 Hz, ⁴J = 2.8 Hz, 1H, Ph), 7.32 (d, J = 2.8 Hz, 1H, Ph), 7.45 (d, J = 9.0 Hz, 1H, Ph), 10.60 (s, 1H, NH⁺). LC/MS: m/z calc. 548.23, found 548.45. Anal. calcd. for C₂₅H₃₅Cl₂N₇O₂···HCl: C, H, N.

1,3-Dimethyl-7-[5-(4-phenylpiperazin-1-yl)pentyl]-8-(1-piperidyl)-purine-2,6-dione hydrochloride (33)

Yield 58%, m.p. 229–230 °C, R_f = 0.45 (A), ¹H-NMR (DMSO) δ: 1.18–1.25 (m, 2H, N7-(CH₂)₂CH₂), 1.55–1.79 (m, 10H, N7-CH₂CH₂CH₂CH₂, N(CH₂)₃ piperidine), 3.06–3.18 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.43–3.52 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.78–3.87 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.06 (t, J = 6.9 Hz, 2H, N7-CH₂,), 6.84 (t, J = 7.0 Hz, 1H, Ph), 6.98 (d, J = 6.0 Hz, 2H, Ph), 7.25 (t, J = 8.0 Hz, 2H, Ph), 10.61 (s, 1H, NH⁺). LC/MS: m/z calc. 494.32, found 494.54. Anal. calcd. for C₂₇H₃₉N₇O₂···HCl: C, H, N.

7-{5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (34)

Yield 56%, m.p. 198–199 °C, R_f = 0.39 (A), ¹H-NMR (DMSO) δ: 1.18–1.27 (m, 2H, N7-(CH₂)₂CH₂), 1.55–1.79 (m, 10H, N7-CH₂CH₂CH₂CH₂, N(CH₂)₃ piperidine), 3.01–3.20 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂-piperidine), 3.19 (s, 3H, N1-CH₃), 3,36 (s, 3H, N3-CH₃), 3.41–3.55 (m, 4H, NH⁺(CH₂CH₂)₂N), 3.76 (s, 3H, OCH₃), 4.06 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.86–7.00 (m, 4H, Ph), 10.15 (s, 1H, NH⁺). LC/MS: m/z calc. 524.33, found 524.46. Anal. calcd. for C₂₈H₄₁N₇O₃···HCl: C, H, N.

7-{5-[4-(3,4-Dichlorophenyl)piperazin-1-yl]pentyl}-1,3-dimethyl-8-(1-piperidyl)-purine-2,6-dione hydrochloride (35)

Yield 62%, m.p. 243–244 °C, R_f = 0.50 (A), ¹H-NMR (DMSO) δ: 1.19–1.30 (m, 2H, N7-(CH₂)₂CH₂(CH₂)₂), 1.55–1.79 (m, 10H, N7-CH₂CH₂CH₂CH₂, N(CH₂)₃ piperidine), 3.11–3.19 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂)₂ piperidine), 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃)_, 3.39–3.45 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.55–3.60 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.05 (t, J = 7.1 Hz, 2H, N7-CH₂), 7.18–7.20 (m, 1H, Ph), 7.32–7.40 (m, 2H, Ph), 10.59 (s, 1H, NH⁺). LC/MS: m/z calc. 562.24, found 562.48. Anal. calcd. for C₂₇H₃₇Cl₂N₇O₂···HCl: C, H, N.

7-{4-[4-(2-Fluorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-morpholino-purine-2,6-dione hydrochloride (36)

Yield 69%, m.p. 210–212 °C, R_f = 0.17 (A), ¹H-NMR (DMSO-d₆) δ: 1.67–1.81 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.04–3.19 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂CH₂)₂O), 3.20 (s, 3H, N1-CH₃), 3.38 (s, 3H, N3-CH₃), 3.48–3.68 (m, 4H, N(CH₂CH₂)₂O), 3.72–3.75 (m, 4H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 4.09–4.14 (m, 2H, N7-CH₂), 6.99–7.10 (m, 4H, Ph), 10.60 (s, 1H, NH⁺). LC/MS: m/z calc. 500.27, found 500.60. Anal. calcd. for C₂₅H₃₄ClN₇O₃···HCl: C, H, N.

7-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}-1,3-dimethyl-8-morpholino-purine-2,6-dione hydrochloride (37)

Yield 66%, m.p. 209–211°C, R_f = 0.35 (A), ¹H-NMR (DMSO-d₆) δ: 1.67–1.78 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.09–3.17 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂CH₂)₂O), 3.18 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.41–3.47 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 3.71–3.74 (m, 4H, N(CH₂CH₂)₂O), 3.83–3.87 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 4.09 (t, J = 6.4 Hz, 2H, N7-CH₂), 6.97 (dd, ³J = 8.9 Hz, ⁴J = 2.8 Hz, 1H, Ph), 7.20 (d, ⁴J = 2.8 Hz, 1H, Ph), 7.42 (d, ³J = 8.9 Hz, 1H, Ph), 10.85 (s, 1H, NH⁺). LC/MS: m/z calc. 550.20, found 550.40. Anal. calcd. for C₂₅H₃₃Cl₂N₇O₃···HCl: C, H, N.

7-{5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentyl}-1,3-dimethyl-8-morpholino-purine-2,6-dione hydrochloride (38)

Yield 62%, m.p. 207–209 °C, R_f = 0.16 (A), ¹H-NMR (DMSO) δ: 1.19–1.26 (m, 2H, N7-(CH₂)₂CH₂), 1.68–1.79 (m, 4H, N7-CH₂CH₂CH₂CH₂), 2.98–3.25 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, N(CH₂CH₂)₂O), 3.20 (s, 3H, N1-CH₃), 3.37 (s, 3H, N3-CH₃), 3.44–3.48 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 3.72–3.75 m, 4H, N(CH₂CH₂)₂O), 3.76 (s, 3H, OCH₃), 4.06–4.14 (m, 2H, N7-CH₂), 6.85–7.00 (m, 4H, Ph), 10.58 (s, 1H, NH⁺). LC/MS: m/z calc. 526.31, found 526.06. Anal. calcd. for C₂₇H₃₉N₇O₄···HCl: C, H, N.

8-(Diethylamino)-7-{3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}-1,3-dimethyl-purine-2,6-dione hydrochloride (39)

Yield 59%, m.p. 203–205 °C, R_f = 0.40 (A), ¹H-NMR (DMSO-d₆) δ: 1.09 (t, J = 7.1 Hz, 6H, N(CH₂CH₃)₂), 2.16–2.27 (m, 2H, N7-CH₂CH₂CH₂), 2.93–3.08 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.20 (s, 3H, N1-CH₃), 3.22–3.28 (m, 4H, N(CH₂CH₃)₂), 3.39 (s, 3H, N3-CH₃), 3.33–3.48 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 3.76 (s, 3H, OCH₃), 4.12 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.87–7.04 (m, 4H, Ph), 10.20 (s, 1H, NH⁺). LC/MS: m/z calc. 484.30, found 484.52. Anal. calcd. for C₂₅H₃₇N₇O₃···HCl: C, H, N.

7-{3-[4-(3,4-Dichlorophenyl)piperazin-1-yl]propyl}-8-(diethylamino)-1,3-dimethyl-purine-2,6-dione hydrochloride (40)

Yield 61%, m.p. 264–266°C, R_f = 0.68 (A), ¹H-NMR (DMSO-d₆) δ: 1.08 (t, J = 7.1 Hz, 6H, N(CH₂CH₃)₂), 2.06–2.20 (m, 2H, N7-CH₂CH₂CH₂), 3.00–3.10 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.12–3.16 (m, 4H, N(CH₂CH₃)₂), 3.20 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃). 3.86–4.14 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 4.11 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.95–6.99 (dd, ³J = 8.9 Hz, ⁴J = 5.9 Hz, 1H, Ph), 7.21 (d, J = 2.8 Hz, 1H, Ph), 7.42–7.44 (d, J = 8.9 Hz, 1H, Ph), 10.20 (s, 1H, NH⁺). LC/MS: m/z calc. 522.21, found 522.48. Anal. calcd. for C₂₄H₃₃Cl₂N₇O₂···HCl: C, H, N.

8-(Diethylamino)-7-{4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl}-1,3-dimethyl-purine-2,6-dione hydrochloride (41)

Yield 72%, m.p. 224–226°C, R_f = 0.24 (A), ¹H-NMR (DMSO-d₆) δ: 1.08 (t, J = 6.9 Hz, 6H, N(CH₂CH₃)₂), 1.60–1.80 (m, 4H, N7-CH₂CH₂CH₂), 3.07–3.14 (m, 6H, CH₂NH⁺(CH₂CH₂)₂N-Ph), 3.19 (s, 3H, N1-CH₃) 3.21–3.28 (m, 4H, N(CH₂CH₃)₂), 3.36 (s, 3H, N3-CH₃), 3.44–3.46 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 3.76 (s, 3H, OCH₃), 4.08 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.85–7.03 (m, 4H, Ph), 11,06 (s, 1H, NH⁺). LC/MS: m/z calc. 498.31, found 498.43. Anal. calcd. for C₂₆H₃₉N₇O₃···HCl: C, H, N.

7-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}-8-(diethylamino)-1,3-dimethyl-purine-2,6-dione hydrochloride (42)

Yield 69%, m.p. 219–221°C, R_f = 0.24 (A), ¹H-NMR (DMSO-d₆) δ: 1.07 (t, J = 6.9 Hz, 6H, N(CH₂CH₃)₂), 1.60–1.71 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.03–3.14 (m, 6H, CH₂NH⁺(CH₂CH₂)₂), 3.18 (s, 3H, N1-CH₃), 3.20–3.27 (m, 4H, N(CH₂CH₃)₂), 3.35 (s, 3H, N3-CH₃), 3.42–4.04 (m, 4H, NH⁺(CH₂CH₂)₂), 4.07 (t, J = 7.1 Hz, 2H, N7-CH₂), 6.95–6.99 (d, J = 8.9, 1H, Ph), 7.20 (s, 1H, Ph), 7.40–7.43 (d, 1H, Ph), 11.30 (s, 1H, NH⁺). LC/MS: m/z calc. 536.23, found 536.38. Anal. calcd. for C₂₅H₃₅Cl₂N₇O₂···HCl: C, H, N.

8-(Dipropylamino)-7-{3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}-1,3-dimethyl-purine-2,6-dione hydrochloride (43)

Yield 75%, m.p. 203–205 °C, R_f = 0.22 (A), ¹H-NMR (DMSO-d₆) δ: 0.85 (t, J = 7.4 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.45–1.58 (m, 4H, N(CH₂CH₂CH₃)₂), 2.15–2.25 (m, 2H, N7-CH₂CH₂CH₂); 3.05–3.60 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N, C(8)N(CH₂)₂), 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.76 (s, 3H, OCH₃), 3.86–4.04 (m, 4H, NH⁺(CH₂CH₂)₂), 4.13 (t, J = 7.4 Hz, 2H, N7-CH₂), 6.84–6.99 (m, 4H, Ph), 11.00 (s, 1H, NH⁺). LC/MS: m/z calc. 512.33, found 512.47. Anal. calcd. for C₂₇H₄₁N₇O₃···HCl: C, H, N.

7-{3-[4-(3,4-Dichlorophenyl)piperazin-1-yl]propyl}-8-(dipropylamino)-1,3-dimethyl-purine-2,6-dione hydrochloride (44)

Yield 67%, m.p. 246–249 °C, R_f = 0.34 (A), ¹H-NMR (DMSO-d₆) δ: 0.84 (t, J = 7,4 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.45–1.57 (m, 4H, N(CH₂CH₂CH₃)₂), 2.17–2.22 (m, 2H, N7-CH₂CH₂CH₂), 3.00–3.20 (m, 10H, NH⁺(CH₂CH₂)₂N, C(8)N(CH₂)₂) 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.47–3.50 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.86–3.89 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.12 (t, J = 7.4 Hz, 2H, N7-CH₂), 6.99–7.05 (d, 1H, Ph), 7.21 (s, 1H, Ph), 7.41–7.44 (d, 1H, Ph), 10.66 (s, 1H, NH⁺). LC/MS: m/z calc. 550.24, found 550.36. Anal. calcd. for C₂₆H₃₇Cl₂N₇O₂···HCl: C, H, N.

8-(Dipropylamino)-7-{4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl}-1,3-dimethyl-purine-2,6-dione hydrochloride (45)

Yield 67%, m.p. 185–186 °C, R_f = 0.22 (A), ¹H-NMR (DMSO-d₆) δ: 0.84 (t, J = 7.1 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.44–1.54 (m, 4H, N(CH₂CH₂CH₃)₂), 1.60–1.71 (m, 4H, N7-CH₂CH₂CH₂CH₂), 2.97–3.11 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, C(8)N(CH₂)₂), 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.44–3.47 (m, 4H, NH⁺(CH₂CH₂)₂N-Ph), 3.76 (s, 3H, OCH₃), 4.07 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.87–6.99 (m, 4H, Ph), 10.68 (s, 1H, NH⁺). LC/MS: m/z calc. 526.35, found 526.51. Anal. calcd. for C₂₈H₄₃N₇O₃···HCl: C, H, N.

7-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-8-(dipropylamino)-1,3-dimethyl-purine-2,6-dione hydrochloride (46)

Yield: 59%, m.p.: 138–140 °C, R_f = 0.46 (A), ¹H-NMR (DMSO-d₆) δ: 0.83 (t, J = 7.4 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.43–1.55 (m, 4H, N(CH₂CH₂CH₃)₂), 1.60–1.71 (m, 4H, N7-CH₂CH₂CH₂CH₂), 2.97–3.18 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, C(8)N(CH₂)₂), 3.19 (s, 3H, N1-CH₃), 3.36 (s, 3H, N3-CH₃), 3.44–3.47 (m, 2H, NH⁺(CH₂CH₂)₂N-Ph), 3.51–3.57 (m, 2H, (CH₂CH₂)₂N-Ph), 4.07 (t, J = 6.9 Hz, 2H, N7-CH₂), 6.85 (dd, ³J = 7.7 Hz, ⁴J = 1.5 Hz, 1H, Ph); 6.93 (dd, ³J = 8.2 Hz, ⁴J = 2.0 Hz, 1H, Ph), 7.02 (t, J = 2.3 Hz, 1H, Ph), 7.23 (t, J = 8.2 Hz, 1H, Ph), 10.42 (s, 1H, NH⁺). LC/MS: m/z calc. 530.30, found 530.47. Anal. calcd. for C₂₇H₄₀ClN₇O₂···HCl: C, H, N.

7-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}-8-(dipropylamino)-1,3-dimethyl-purine-2,6-dione hydrochloride (47)

Yield: 68%, mp: 169–171 °C, R_f = 0.28 (A), ¹H-NMR (DMSO-d₆) δ: 0.84 (t, J = 6.9 Hz, 6H, N(CH₂CH₂CH₃)₂), 1.44–1.56 (m, 4H, N(CH₂CH₂CH₃)₂), 1.70–1.72 (m, 4H, N7-CH₂CH₂CH₂CH₂), 3.01–3.15 (m, 10H, CH₂NH⁺(CH₂CH₂)₂N-Ph, C(8)N(CH₂)₂), 3.18 (s, 3H, N1-CH₃), 3.35 (s, 3H, N3-CH₃), 3.43–3.47 (m, 2H, NH⁺(CH₂CH₂)₂N), 3.84–3.88 (m, 2H, NH⁺(CH₂CH₂)₂N), 4.07 (t, J = 7.1 Hz, 2H, N7-CH₂), 7.06 (dd, ³J = 8.0 Hz, ⁴J = 2.8 Hz, 1H, Ph), 7.22 (d, J = 2.8 Hz, 1H, Ph), 7.45 (d, J = 9.0 Hz, 1H, Ph), 10.81 (s, 1H, NH⁺). LC/MS: m/z calc. 564.26, found 564.40. Anal. calcd. for C₂₇H₃₉Cl₂N₇O₂···HCl: C, H, N.

X-ray structure determination and refinement

Good quality single crystals of the representative compounds 37 and 44 were obtained from ethanol solution by slow evaporation of the solvent at ambient condition. Few drops of water were added in order to increase solubility. X-ray diffraction data were collected at 120(2) K using SuperNova diffractometer (Agilent Technologies, Inc., Santa Clara, CA) with MoKα radiation (λ = 0.71073 Å) and processed with CRYSALIS^Pro27. The phase problem was solved by direct methods with SHELXS-97 (Agilent Technologies, Inc., Santa Clara, CA)28. Parameters of the obtained model were refined by full-matrix least-squares on F² using SHELXL-97 (Agilent Technologies, Inc., Santa Clara, CA)28. All non-hydrogen atoms were refined anisotropically. H atoms attached to nitrogen atom N15 and these in water molecule were found on the difference Fourier map.

Positions of hydrogen atoms attached to carbon atoms were calculated with C–H = 0.95 Å for aromatic, C–H = 0.99 Å for methylene, C–H = 0.98 Å for methyl groups and were refined using the riding model with the isotropic displacement parameter U_iso[H] = 1.2 U_eq[C] or U_iso[H] = 1.5 U_eq[C] (the last one constraint for the methyl groups only).

All crystallographic data for compounds 37 and 44 presented structures are shown in Supplemental Information\. WinGX29 software (Gaussian Inc., Pittsburgh, PA) was used to prepare materials for publication. Figure 2 shows asymmetric units of presented structures which were obtained with ORTEP-3 for Windows30.

Figure 2. Asymmetric units of crystal structures of 37 (left panel) and 44 (right panel), showing the atom labeling scheme. Dashed lines represent a charge-assisted hydrogen bond N+-H … Cl- and hydrogen bonds formed by water molecule within the asymmetric unit. Displacement ellipsoids of non-hydrogen atoms are drawn at the 30% probability level. H atoms are presented as small spheres with an arbitrary radius.

Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Center as supplementary publication nos. CCDC 1049600 (37), CCDC 1049601 (44). Copies of the data can be obtained, free of charge, by application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: [phone] or E-mail: [email protected]).

In vitro radioligand binding assays

The compounds 18–47 were tested in competition binding experiments by displacement of respective radioligands from cloned human Rs, all stably expressed in HEK-293 cells: [³H]-8-OH-DPAT, [³H]-ketanserin), [³H]-LSD, [³H]-5-CT, and [³H]-raclopride for 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and dopamine D₂ Rs, respectively according to the previously published procedures8^,31^,32. Each compound was tested in triplicate at 7–8 concentrations (10⁻¹¹–10⁻⁴M). The inhibition constants (K_i) were calculated based on the method described in the literature33. Results were expressed as means of at least two separate experiments.

Functional in vitro evaluation

The tested and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM. Serial dilutions were prepared in 96-well microplate in assay buffers and 8–10 concentrations were tested.

A cellular aequorin-based functional assay was performed with recombinant CHO-K1 cells expressing mitochondrially targeted aequorin, human GPCR, and the promiscuous G protein α16 for 5-HT₆ and 5-HT_2A receptors, Gαqi/5 for D₂ receptor (PerkinElmer Health Sciences, Inc., Shelton, CT). Assay was executed according to previously described protocol11. After thawing, cells were transferred to assay buffer (DMEM/HAM’s F12 with 0.1% proteasefree BSA) and centrifuged. The cell pellet was resuspended in assay buffer and coelenterazine h was added at final concentrations of 5 μM. The cell suspension was incubated at 16 °C, protected from light with constant agitation for 16 h and then diluted with assay buffer to a concentration of 12 500 cells/ml for 5HT₆ and 5HT_2A receptors and 3000 cells/ml for D₂ receptor. After 1 h of incubation, 50 μl of the cell suspension was dispensed using automatic injectors built into the radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer Health Sciences, Inc., Shelton, CT) into white opaque 96-well microplates preloaded with test compounds. Immediate light emission generated following calcium mobilization was recorded for 30−60 s. In antagonist mode, after 15−30 min of incubation the reference agonist was added to the above assay mix and light emission was recorded again. A final concentration of the reference agonist was equal to an EC₈₀ value of 40 nM serotonin for the 5-HT₆ receptor, 30 nM α-methylserotonin for the 5-HT_2A receptor, and 30 nM apomorphine for the D₂ receptor.

For the 5-HT₇ and 5-HT_1A receptors, adenylyl cyclase activity was monitored using cryopreserved CHO-K1 cells, expressing the human serotonin 5-HT₇ or 5-HT_1A receptor. Thawed cells were resuspended in stimulation buffer (HBSS, 5 mM HEPES, 0.5 IBMX, and 0.1% BSA at pH 7.4) at 2000 cells/ml for 5-HT₇ receptor and 4000 cells/ml for 5-HT_1A receptor. Ten microliters of cell suspension was added to 10 μl of tested compounds for 5-HT₇ receptor and 10 μl of tested compounds with 10 µM forskolin for 5-HT_1A receptor loaded onto a white opaque half area 96-well microplate.

An antagonist response experiment was performed with 10 nM serotonin as the reference agonist for 5-HT₇ receptor and 0.7 nM 5-carboxamidotryptamine as the reference agonist for 5-HT_1A receptor. The agonist and the antagonist were added simultaneously. Cell stimulation was performed for 60 min at room temperature. After incubation, cAMP measurements were performed with homogeneous TR-FRET immunoassay using the LANCE Ultra cAMP kit (PerkinElmer Health Sciences, Inc., Shelton, CT). Ten microlites of EucAMP Tracer Working Solution and 10 μl of ULight-anti-cAMP Tracer Working Solution were added, mixed, and incubated for 1 h. The TR-FRET signal was read on an EnVision microplate reader (PerkinElmer Health Sciences, Inc., Shelton, CT). IC₅₀ and EC₅₀ values were determined by non-linear regression analysis using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA).

Molecular modeling

The homology models of human D₂ dopamine and 5-HT_1A serotonin receptors, which were engaged in docking studies, have been described in previously published papers11^,34^,35. The detailed comparative modeling method and ligand-based optimization characteristics developed for this purpose have also been presented formerly36.

The D₂ receptor models were built using dopamine D₃ receptor crystal structure as a template (Protein Data Bank (PDB) database ID: 3PBL)37 while the 5-HT_1A receptor models were built on the basis of β₂ adrenergic receptor crystal structure (PDB ID: 2RH1)38. Sequence alignments between D₂ and 5-HT_1A receptors (UniProt database accession numbers P14416 and P08908, respectively)39 and their templates were due to hhsearch tool via GeneSilico Metaserver40. The crude receptor models were obtained using SwissModel41, and were validated by processing in Protein Preparation Wizard42. For each receptor type, a set of bioactive compounds was selected for ligand-steered binding site optimization, which was completed using induced fit docking (IFD) workflow43^,44. That procedure resulted in a variety of receptor models that served as molecular targets in docking studies, mimicking conformational freedom of the proteins.

Ligand structures were optimized using LigPrep tool. Glide XP flexible docking was carried out using default parameters, leaving ring conformations as defined during ligand preparation. H-bond constraints, as well as centroid of a grid box (22 × 22 × 22 Å) for docking studies were located on Asp3.32.

Glide, induced fit docking, LigPrep, and Protein Preparation Wizard were implemented in Small-Molecule Drug Discovery Suite (Schrödinger, Inc., New York, NY), which was licensed for Jagiellonian University Medical College.

Behavioral evaluation

The experiments were performed on male Albino Swiss or CD-1 mice (22–28 g). The animals were kept at a room temperature of 20 ± 1 °C, and had free access to food (standard laboratory pellets) and tap water before the experiment. All the experiments were conducted in the light phase between 9 a.m. and 2 p.m. All the experimental procedures were approved by the Local Ethics Commission for Animal Experiments of Jagiellonian University in Cracow. d-Amphetamine (Sigma-Aldrich, St. Louis, MO) and the investigated compounds (32 and 34) were dissolved in distilled water. With the exception of d-amphetamine that was administered subcutaneously (sc), the remaining compounds were injected intraperitoneally (ip). The investigated compounds were administered 60 min and d-amphetamine 30 min before the test. Each experimental group consisted of 6–10 animals, and all the animals were used only once.

d-Amphetamine-induced hyperactivity in CD-1 mice

Locomotor activity was recorded with an Opto M3 multi-channel activity monitor (MultiDevice Software v.1.3, Columbus Instruments, Columbus, OH). The mice were individually placed in plastic cages (22 × 12 × 13 cm) for 30 min habituation period, and then the crossings of each channel (ambulation) were counted during 1 h with data recording every 5 min. The cages were cleaned up with 70% ethanol after each mouse.

Spontaneous locomotor activity in CD-1 mice

Locomotor activity was recorded with an Opto M3 multi-channel activity monitor (MultiDevice Software v.1.3, Columbus Instruments, Columbus, OH). The CD-1 mice were individually placed in plastic cages (22 × 12 × 13 cm) for 30 min habituation period, and then the crossings of each channel (ambulation) were measured every 5 min for 60 min. The cages were cleaned up with 70% ethanol after each mouse.

Results and discussion

Comparative structural analysis

The X-ray crystal structure analysis was performed for 3,4-dichlorophenylpiperazine-purine-2,6-dione derivatives 37 and 44 in order to examine the molecular conformation and intermolecular interactions. The impact of the different amino substituent in the 8 position of purine-2,6-dione scaffold (morpholine and dipropylamine, respectively) and the length of the aliphatic linker (four and three methylene groups, respectively) were investigated.

Compounds 37 and 44 crystallise in the form of hydrochloride salt, with positive charge localized on N15 atom of the piperazine ring. Due to the presence of the cation, the charge-assisted hydrogen bond N15⁺–H15···Cl1⁻ is formed in the crystal structure of 37 and 44. The co-crystallising water molecule additionally forms strong hydrogen bonds as a donor (O–H···Cl⁻ and O−H···O) and as an acceptor of the weak C–H···O interactions (Figure 2 and Supplemental Information).

There are two main differences in the molecular structures of both compounds with a great impact on the crystal packing. First is the length of the aliphatic linker, which consists of three and four methylene groups for 37 and 44, respectively. This linker adopts different conformation in both presented crystal structures. Conformation of linker and its influence on the biological activity was widely discussed in the literature18^,45–48. In most known LCAP crystal structures with tetramethylene linker, this aliphatic chain adopts extended conformation (anti–anti–anti)18. Such conformation is also observed from 37, with torsion angles varying around 170°. However, in the shorter linker of 44, the conformation of the aliphatic trimethylene chain is anti-gauche, with torsion angles N7–C10–C11–C12 and C10–C11–C12–N15 being 178.3° and −70.8°, respectively.

In the molecule of the 37, the polar morpholine ring in the 8 position formed intermolecular interactions of C–H···O type, both as donor and acceptor of the hydrogen bond (Supplemental Information). The same type of interactions was observed in the previously reported 3-Cl and 2-OCH₃ PhP analogs18.

The bulky and hydrophobic dipropylamine substituent in the structure of compound 44 avoids polar intermolecular contacts, forming the hydrophobic planes normal to [001] direction. This hydrophobic association leads to such molecular arrangement which allow antiparallel stacking interaction. The 3,4-dichlorophenyl overlap alternately with the six-membered ring of the purine system (centroid–centroid distance in the range of 3.5–3.6 Å). This π–π interaction propagates continuously along [100] axis.

In vitro binding experiments

Numerous newly synthesized derivatives of 8-amine-purine-2,6-dione derivatives of LCAPs (18–47) displayed high-to-moderate affinity for 5-HT_1A, 5-HT_2A 5-HT_7, and D₂ Rs (K_i = 1–115 nM, 24–106 nM, 26–97 nM, and 2–78 nM, respectively). Moreover, several compounds possessed marked affinity (K_i < 100 nM) for 5-HT₆ Rs (Tables 1 and 2). The 8-piperidine-purine-2,6-dione derivatives (26–35) were interesting because of their multireceptor (serotonin and dopamine) affinities. It seems that this cyclic amine system was preferable to obtain potent serotonin and D₂ ligands. It was previously found in a series of 8-unsubstituted 7-phenylpiperazinylalkyl-purine-2,6-diones that the lack of substituent in a 8 position was rather preferential for the interaction with 5-HT_1A Rs19. In this group of 8-piperidine analogs, the elongation of propylene spacer to butylene one increased the affinities for all evaluated receptors. This modification resulted in 273-fold higher affinities for 5-HT₆, 148-fold for 5-HT₇, and 34-fold for D₂ Rs in the case of 2-OCH₃ derivative 27 (yielding 30). Within a set of structural analogs, 3,4-diCl-PhP derivative (32) possessed high receptor affinities for the targeted receptors, similar to aripiprazole, except 5-HT_1A sites to which was less active. This result may suggest that the 3,4-diCl substituent was preferable to obtain potent 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ ligands (Table 1). Compound 31 containing 3-Cl substituent displayed higher affinity for 5-HT_1A than 32 but was three-fold less active for 5-HT₆ and over six-fold less active for D₂ Rs. The elongation of the linker length between purine-2,6-dione core and PhP fragment from four- to five-carbon units increased threefold D₂ affinity (29 and 30 versus 33 and 34) and 1.5–5 fold the 5-HT_1A affinity (e.g. 29–31 versus 33–35). At the same time, it decreased 5-HT₆ Rs affinity and either retained or slightly decreased the affinity for 5-HT_2A Rs (Table 1).

Table 1. The binding data of the 8-pyrrolidino-, 8-piperidino-, and 8-morpholino-purine-2,6-dione derivatives for 5-HT and dopamine receptors.

				Ki ± SEM [nM]
Compd	R	X	n	5-HT1A	5-HT2A	5-HT6	5-HT7	D2
18	H	–	1	693 ± 73	90 ± 11	>10 000	197 ± 19	973 ± 109
19	2-OCH3	–	1	143 ± 12	435 ± 47	>20 000	285 ± 29	78 ± 8
20	3-Cl	–	1	99 ± 11	67 ± 8	>20 000	120 ± 11	375 ± 38
21	4-F	–	1	1703 ± 183	90 ± 8	7353 ± 905	83 ± 10	1214 ± 127
22	H	–	2	228 ± 19	124 ± 12	>10 000	289 ± 31	136 ± 17
23	2-OCH3	–	2	113 ± 10	145 ± 15	5607 ± 717	89 ± 9	38 ± 5
24	3-Cl	–	2	169 ± 18	436 ± 51	127 ± 12	53 ± 7	24 ± 3
25	4-F	–	2	686 ± 84	106 ± 14	5035 ± 613	434 ± 46	175 ± 19
26	H	CH2	1	27 ± 3	124 ± 11	>10 000	459 ± 58	64 ± 9
27	2-OCH3	CH2	1	27 ± 2	179 ± 16	>20 000	639 ± 70	120 ± 11
28	3,4-diCl	CH2	1	359 ± 43	36 ± 4	>10 000	>10 000	172 ± 16
29	H	CH2	2	115 ± 13	14 ± 3	2034 ± 179	288 ± 29	71 ± 8
30	2-OCH3	CH2	2	12 ± 2	70 ± 6	2865 ± 312	145 ± 13	18 ± 7
31	3-Cl	CH2	2	55 ± 6	40 ± 3	218 ± 19	97 ± 10	32 ± 4
32	3,4-diCl	CH2	2	174 ± 19	36 ± 4	72 ± 8	73 ± 8	5 ± 1
33	H	CH2	3	59 ± 5	31 ± 2	3467 ± 368	62 ± 7	26 ± 3
34	2-OCH3	CH2	3	8 ± 1	47 ± 6	6367 ± 813	26 ± 3	3 ± 1
35	3,4-diCl	CH2	3	35	159 ± 19	275 ± 41	39 ± 4	2 ± 0.2
36	2-F	O	2	255 ± 18	322 ± 41	2447 ± 219	2067 ± 242	211 ± 17
37	3,4-diCl	O	2	408 ± 38	25 ± 3	84 ± 9	118 ± 9	137 ± 17
38	2-OCH3	O	3	103 ± 12	412 ± 33	4960 ± 612	204 ± 18	21 ± 2
Aripiprazole*				5.6 ± 0.8	21 ± 1	90 ± 1	26 ± 2	0.8 ± 0.1
Buspirone				20 ± 2	–	–	–	–
Olanzapine				–	4.6 ± 0.9	7 ± 0.8	–	7 ± 0.6
Clozapine				–	–	–	18 ± 2	–

*Data taken from literature49.

Table 2. The binding data of the 8-diethylamino- and 8-dipropylamino-purine-2,6-dione derivatives for 5-HT and dopamine receptors.

			Ki ± SEM (nM)
Compd	R	n	5-HT1A	5-HT2A	5-HT6	5-HT7	D2
39	2-OCH3	1	11 ± 2	192 ± 21	>20 000	118 ± 12	11 ± 1
40	3,4-diCl	1	58 ± 6	347 ± 38	372 ± 41	233 ± 19	63 ± 7
41	2-OCH3	2	64 ± 8	566 ± 63	>20 000	577 ± 63	59 ± 5
42	3,4-diCl	2	234 ± 27	285 ± 32	>10 000	461 ± 49	534 ± 47
43	2-OCH3	1	17 ± 2	249 ± 28	688 ± 87	130 ± 15	11 ± 2
44	3,4-diCl	1	162 ± 21	240 ± 31	94 ± 12	402 ± 52	59 ± 7
45	2-OCH3	2	16 ± 2	27 ± 4	42 ± 5	83 ± 9	60 ± 8
46	3-Cl	2	48 ± 6	199 ± 24	90 ± 11	53 ± 6	57 ± 9
47	3,4-diCl	2	<1	84 ± 11	260 ± 30	122 ± 14	20 ± 3
Aripiprazole			5.6 ± 0.8	21 ± 1	90±	26 ± 2	0.8 ± 0.1
Buspirone			20 ± 2	–	–	–	–
Olanzapine			–	4.6 ± 0.9	7 ± 0.8	–	7 ± 0.6
Clozapine			–	–	–	18 ± 2	–

Hence, in the tested series, the four-methylene group spacer was preferable to obtain multifunctional receptor ligands. The replacement of the piperidine substituent with morpholine moiety with additional hydrogen bond acceptor generally decreased the affinity for the evaluated receptors as was previously evidenced in a series of 7-arylpiperazinylalkyl-8-morpholin-4-yl-purine-2,6-dione derivatives18.

The 8-morpholine-3,4-diCl-PhP analog of 32, compound 37, explained similar 5-HT_1A, 5-HT₂, and 5-HT₆ receptor affinity but was less active for 5-HT₇ and D₂ Rs. The elongation of the alkyl chain to five-carbon unit in a series of 8-morpholin-4-yl-purine-2,6-diones did not significantly change the affinity for the evaluated receptors in comparison with the previously reported analogs of the four aliphatic linker18.

Compound 38 with 2-OCH₃ substituent and five methylene spacer was three-fold and 14-fold less active at 5-HT_1A sites in comparison with the previously reported analogs with three-carbon and four-carbon linkers, respectively18. At the same time, this modification retained its activity for evaluated 5-HT_2A, 5-HT₆, and 5-HT₇ receptors.

The introduction of 3,4-diCl substituent into the PhP moiety significantly increased the affinity for 5-HT₆ receptors. This effect was observed in the case of 32 and 37 among four-methylene analogs with piperidine (32) or morpholine (37) moiety in a 8-position of purine-2,6-dione core. Furthermore, this substituent in the PhP fragment is also preferable for D₂ receptors. The reduction of cyclic amine system from six-membered piperidine to pyrrolidine ring generally decreased 5-HT_1A, 5-HT_2A, and D₂ affinities but increased up to two-fold 5-HT₇ affinity. Compound 24 with 3-Cl substituted phenylpiperazine displayed high affinity for 5-HT₇ and D₂ sites and moderate affinity to other evaluated receptors.

Generally, compounds with a dipropylamine substituent in an 8-position (43–47) may be classified as multimodal serotonin and dopamine receptor ligands with high-to-moderate affinity for 5-HT_1A, 5-HT_2A, 5-HT_6, 5-HT₇, and D₂ receptors (Table 2). This modification was significantly beneficial for 5-HT₆ affinity. Compound 45 was 68-fold more active than its 8-piperidinyl counterpart 30. This may suggest that the hydrophobic substituent in a 8-position of purine-2,6-dione core may account for higher affinity for 5-HT₆ sites. The replacement of the dipropylamine group by diethylamine moiety significantly reduced 5-HT₆ affinity. The impact of this substituent for the affinities for other targeted receptors was not so decisive. Summing up, the binding study allowed us to identify some multimodal 5-HT/D receptor ligands. Among them, the most interesting compounds were selected for the determination of their functional profile.

Functional in vitro evaluation

On the basis of the multireceptor binding affinity results, a series of 11 compounds was selected for the functional profile characterization in vitro at all five targets, according to the previously published methods11. Potent D₂R antagonists were identified among the selected 8-aminopurine-2,6-dione derivatives, achieving single-digit nanomolar IC₅₀ values (34, 41, and 45) and, in this way, were comparable with the chlorpromazine (Table 3). Other evaluated compounds (24, 31, 32, 39, and 46) displayed antagonistic properties at this receptor with double-digit nanomolar IC₅₀ values. In addition, 43 and 47 showed antagonistic activity with IC₅₀ values > 100 nM. On the contrary, the majority of the evaluated compounds showed agonistic activity at D₂R, achieving double-digit nanomolar value in the case of 34 and 45 (Table 3). Regarding the functional profile the tested compounds are similar to aripiprazole (partial agonist of D₂R) that is in line with the recent trends in the development of modern atypical antipsychotics. Moreover, five from the evaluated compounds (31, 34, 39, 41, and 43) behaved as partial agonists of 5-HT_1A receptors (Table 4). It is well known that 5-HT_1A receptor agonists or partial agonists may attenuate the catalepsy induced by the D₂R blockade, as well as reduce the cataleptogenic potential of novel antipsychotic agents50. In addition, compounds 31, 32, 43, 45, and 46 showed antagonistic properties at 5-HT_2A receptors in a range of IC₅₀ value = 34.6–616 nM, and, at the same time, displayed 5-HT_2A agonistic activity (Tables 4 and 5). The use of 5-HT_2A antagonists in dopamine-deficient areas of the brain, including the frontal and nigrostriatal pathways, may improve the negative symptoms of schizophrenia and decrease the incidence of extrapyramidal side effects51^,52. Among the evaluated compounds, weak 5-HT₇R antagonistic properties were observed in the case of 34 (Table 5). Taking into account the functional profile, the evaluated compounds corresponded to aripiprazole which exerts not only partial dopaminergic activity but also a 5-HT_1A and 5-HT_2C receptor partial agonist and 5-HT_2A and 5-HT₇ antagonist and this mechanism of action may contribute to its broad efficacy – antipsychotic, antidepressant, and anxiolytic53–55.

Table 3. Functional data (agonist and antagonist mode) for the selected compounds for dopamine D₂ receptor.

Compd	EC50 (nM) (pEC50 ± SEM)	IC50 (nM) (pIC50 ± SEM)
24	158 6.80 ± 0.11	41.1 7.39 ± 0.06
31	627 6.20 ± 0.12	36.8 7.43 ± 0.06
32	nc	40.7 7.39 ± 0.10
34	67.3 7.17 ± 0.16	2.97 8.53 ± 0.08
39	219 6.66 ± 0.06	80.8 7.09 ± 0.11
40	nc	6120 5.21 ± 0.30
41	105 6.98 ± 0.09	6.74 8.17 ± 0.09
43	323 6.49 ± 0.09	162 6.79 ± 0.11
45	45.5 7.34 ± 0.11	4.07 8.39 ± 0.10
46	592 6.23 ± 0.07	38.5 7.42 ± 0.08
47	>10 000	591 6.23 ± 0.10
Apomorphine	7.8 8.1 ± 0.09	–
Chlorpromazine	–	1.59 8.80 ± 0.06
Aripiprazole*	8.2	39

nc, not calculable.

*Data taken from literature 58.

Table 4. Functional data (agonist mode) for the selected compounds for serotonin receptors.

	EC50 (nM) (pEC50 ± SEM)
Compd	5-HT1A	5-HT2A	5-HT6	5-HT7
24	nc	nc	nc	5630 5.25 ± 0.93
31	369 6.43 ± 0.27	223 6.65 ± 0.06	nc	1460 5.84 ± 0.20
32	507 6.3 ± 0.19	91.8 7.04 ± 0.04	nc	1540 5.83 ± 0.23
34	15.5 7.81 ± 0.10	nc	nc	1200 5.92 ± 0.28
39	135 6.87 ± 0.07	nt	nt	nt
40	1490 5.83 ± 0.21	nt	nt	nt
41	45.8 7.34 ± 0.11	nt	nt	nt
43	301 6.52 ± 0.35	869 6.06 ± 0.07	nc	–
45	1510 6.82 ± 0.20	nc	nc	3400 5.47 ± 0.38
46	nc	59.3 7.23 ± 0.04	nc	1210 5.92 ± 0.34
47	8840 5.05 ± 0.34	1950 5.71 ± 0.12	nc	7020 5.15 ± 0.72
5-carboxamido-tryptamine	0.65 9.19 ± 0.22
α-methyl-serotonin		1.39 8.86 ± 0.04
serotonin			2.05 8.69 ± 0.07	1.61 8.79 ± 0.06

nc, not calculable; nt, not tested.

Table 5. Functional data (antagonist mode) for the selected compounds for serotonin receptors.

	IC50 (nM) (pIC50 ± SEM)
Compd	5-HT1A	5-HT2A	5-HT6	5-HT7
24	407 6.39 ± 0.30	1490 5.83 ± 0.08	3820 5.42 ± 0.18	5.91 ± 0.07
31	797 6.10 ± 0.47	74.5 7.13 ± 0.11	2580 5.59 ± 0.1	5.4 ± 0.11
32	1050 5.98 ± 0.13	41.8 7.38 ± 0.05	1740 5.76 ± 0.1	–
34	38.3 7.42 ± 0.09	3500 5.46 ± 0.14	nc	6.05 ± 0.05
39	248 6.61 ± 0.09	nt	nt	nt
40	6070 5.22 ± 0.37	nt	nt	nt
41	48.3 7.32 ± 0.09	nt	nt	nt
43	664 6.20 ± 0.16	616 6.21 ± 0.05	676 6.17 ± 0.28	–
45	145 6.84 ± 0.09	318 5.50 ± 0.08	2460 5.61 ± 0.13	–
46	1680 5.78 ± 0.48	34.6 7.46 ± 0.04	847 6.07 ± 0.13	–
47	6850 5.17 ± 0.29	1080 5.97 ± 0.05	1460 5.84 ± 0.17	–
WAY 100135	31.6 7.50 ± 0.07
NAN 190	5.99 8.22 ± 0.12
Pimavanserin		5.7 8.24 ± 0.09
Mianserin			744 6.13 ± 0.12
SB 269970				5.42 8.27 ± 0.11

nc, not calculable; nt, not tested.

Moreover, among the evaluated compounds, 43 and 46 showed 5-HT₆ antagonistic properties (IC₅₀ values = 676 and 847 nM, respectively) (Table 5). It seems important in terms of the recent reports that proposed 5-HT₆ receptor antagonists as potential drugs to treat cognitive impairments in schizophrenia56^,57.

Molecular modeling

Molecular modeling studies have been performed in order to characterize putative binding mode of the bioactive representative of the chemical class of arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione – compound 34. Docking studies extensively facilitated interpretation of SAR data and defined possible structural modifications.

Compound 34 displayed potent affinity for 5-HT_1A receptor, which is of major interest in the presented research. The molecule adopts consistent conformation in the homology models of 5-HT_1A receptor (Figure 3A). The protonated nitrogen atom of the arylpiperazine moiety binds to Asp3.32 (charge-reinforced H-bond), while the aryl ring interacts with Phe6.52 (π–π stacking) and Lys191 (π-cation). Binding mode analysis confirmed that substitution of the methoxy group in position 2 of the aryl ring is the most beneficial in the series, since it enables H bonding with Lys191 and does not interfere with other aminoacid residues constituting the binding pocket59. The 1,3-dimethylpurine-2,6-dione moiety stabilizes the ligand–receptor complex by π–π stacking with Tyr2.64, while its substituent in position 8 fills the non-specific cavity below the second extracellular loop (ECL 2). The latter moiety does not determine affinity for this receptor subtype, but may introduce activity towards other subtypes of receptors, e.g. 5-HT_6, which is evident in the presented series, although further investigation is required to explain it.

Figure 3. Binding modes of compound 34 in the site of 5-HT_1A receptor (A) and D₂ receptor (C). Proposed bioactive conformations and important interactions of compound 34 (green) and aripiprazole (cyan) in the binding site of D₂ receptor (B). Amino acid residues engaged in ligand binding (within 4 A from the ligand atoms) are displayed as sticks, whereas those forming important H-bonds (dotted yellow lines) or π–π stacking/π-cation interactions (dotted green lines) are represented as thick sticks. For the sake of clarity, ECL2 residues were hidden. TMH, transmembrane helix; ECL, extracellular loop.

The present series of compounds display pronounced affinity for dopamine D₂ receptor, which has been secured by introducing privileged arylpiperazine scaffold60. Functional studies revealed that several compounds of the series were characterized as D₂ receptor partial agonists. Analysis of SAR data in this class of ligands suggests that the second terminal part of molecules may be responsible for elicitation of intrinsic activity61^,62. Arylpiperazine part of the analyzed compound 34 interacts with D₂ receptor’s Asp3.32 and Phe6.51/6.52, as described for 5-HT_1A receptor, while 8-piperidine-1,3-dimethylpurine-2,6-dione moiety occupies less conserved cavity, having aromatic contacts with Tyr7.35 and H-bond with Ser7.36 (Figure 3C).

The proposed binding mode resembles the one of aripiprazole, antipsychotic drug, exhibiting well-balanced partial agonism for D₂ receptor63. The docking poses of compound 34 and aripiprazole shown to be very consistent. Besides the arylpiperazine moieties, which took the same position in the orthosteric pocket, the 8-piperidine-1,3-dimethylpurine-2,6-dione and 3,4-dihydroquinolin-2(1H)-one fragments appeared to interact in the same manner, both accepting H-bond from the hydroxy group of Ser7.36 and filling fairly hydrophobic cavity between THMs 2 and 7 (Figure 3B). Such binding site interactions are supposed to cause conformational changes promoting receptor activation (compare in vitro data in Table 3).

The results of the described X-ray studies confirmed the outcomes of structure generation by means of molecular modeling, e.g. chair conformation of piperazine ring which is substituted equatorially. The aliphatic linker adopts extended conformation and the heterocyclic substituent in a 8 position of purine-2,6-dione core preserved the same equatorially substituted chair conformation.

Behavioral evaluation

Both the receptor profile and the functional activity of compounds 32 and 34 prompted us to evaluate potential antipsychotic activity using d-amphetamine-induced hyperactivity in CD-1 mice. d-Amphetamine produces locomotor hyperactivity in animals as a result of increased dopaminergic activity in the mesolimbic system64^,65. There are many evidences that its effect is blocked by antipsychotics having antagonist properties toward dopamine receptors66. The obtained, in our experiment, results indicate that both investigated compounds 32, at dose of 20 mg/kg, and 34, administered at doses of 10 and 20 mg/kg, revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice by 77.5, 80, and 97%, respectively (Table 6).

Table 6. The effect of compounds 32 and 34 on the d-amphetamine-induced locomotor activity in mice.

Compd (dose mg/kg)	Number of movements/60 min
Vehicle + vehicle	2792,0 ± 554,5
Vehicle + AMP(2.5)	9410,6 ± 1833,5*
32 (20) + AMP (2.5)	2114,0 ± 1047,3##
32 (10) + AMP (2.5)	8932,7 ± 1211,2
32 (5) + AMP (2.5)	6500,2 ± 1717,1
32 (2.5) + AMP (2.5)	5507,7 ± 1022,9
	F(5,49) = 4.6961 p < 0.01
Vehicle + vehicle	2131,1 ± 436,3
Vehicle + AMP (2.5)	5387,2 ± 676,7*
34 (20) + AMP (2.5)	155,0 ± 43,5####
34 (10) + AMP (2.5)	1041,3 ± 205,6####
34 (5) + AMP (2.5)	2869,1 ± 641,2
34 (2.5) + AMP (2.5)	3307,6 ± 947,0
	F(5,47) = 8.9043 p < 0.0001

The investigated compounds were administered ip 60 min, and AMP sc 30 min before the test. n = 8–10 mice per group.

*p < 0.05 versus the vehicle + vehicle group, ##p < 0.01, ####p < 0.0001 versus the vehicle + AMP group.

AMP, d-amphetamine.

The effect seems to be specific, since 32 administered at effective dose had no influence on the spontaneous locomotor activity in mice. In contrary, the reduction of d-amphetamine induced hyperactivity caused by compound 34 is probably due to its sedative potential (Supplemental information). The present preclinical results demonstrate that compound 32 has pharmacological characteristics of an antipsychotic agent and encourage to pursue further more advanced studies of this structure.

Conclusion

Extending the studies in a group of 7-arylpipiperazinylalkyl-8-amino-1,3-dimethylpurine-2,6-diones, some potent multireceptor 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands were identified. The 8-piperidine (26–35) and 8-dipropyloamino (45–47) derivatives were interesting because of their multireceptor (serotonin and dopamine) affinities which may indicate that this cyclic amine systems were preferable to obtain potent ligands for evaluated receptors. In these series, the four- and the five-methylene group alkyl spacer was beneficial for binding affinity for targeted receptors. A strong positive influence of 3-chloro- and 3,4-dichloro-phenylpiperazinyl moieties for 5-HT₆ receptor affinity should be pointed out. Compounds 32, 37, and 44–46 were active at these sites similar to aripiprazole. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. In the behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, which in the case of 32 seems to be specific. The obtained results imply that the series of the novel long-chain arylpiperazine derivatives of 8-aminopurine-2,6-dione is worthy of future research for their potential antipsychotic- and/or antidepressant-like activity.

Supplementary material available online

Acknowledgements

This study was supported by the National Science Centre (NCN) - Poland funded Grant No. UMO-2011/01/B/NZ4/00695 and No. 2012/07B/NZ7/01173. The crystallographic research was carried out with the equipment purchased using financial support of the European Regional Development Fund in the framework of the Polish Innovative Economy Operational Program (contract no. POIG.02.01.00-12-023/08).

Declaration of interest

The authors report that they have no conflict of interest.